Steroid 5-.alpha.-reductase is a NADPH-dependent enzyme that converts testosterone to DHT. Recognition of the importance of elevated DHT levels in various disease states has stimulated many efforts to synthesize inhibitors of this enzyme.
The first inhibitor described was 4-androsten-3-one-17.beta.-carboxylic acid by Hsia and Voight in 1973. J. Invest. Dermat, 62:224-227. (4R)-5,10-seco-19-norpregna-4,5-diene -3,10,20-triane was the next inhibitor to be described and also has found utility as an affinity label for 5-.alpha.-reductase. Robaire, B., et al., (1977), J. Steroid Biochem. 8:307-310. (5.alpha.,20-R)-4-diazo-21-hydroxy-20-methylpregnan-3-one has been reported as a potent, time-dependent inhibitor of steroid 5-.alpha.-reductase. Blohm, T. R., et al., (1980), Biochem. Biophys. Res. Comm, 95:273-280; U.S. Pat. No. 4,317,817, Mar. 2, 1982. 17.beta.-N,N-diethylcarbamoyl-4-methyl-4-aza -5-.alpha.-androstan-3-one is exemplary of a group of 4-aza steroid inhibitors of steroid 5-.alpha.-reductase described in U.S. Pat. No. 4,377,584 which issued Mar. 22, 1983, and in Liang, T., et al., (1983), J. Steroid Biochem, 19:385-390. 17.alpha.-acetoxy-6-methylene-pregn-4-ene-3,20 -dione also has been shown to be a time-dependent inactivator of steroid 5-.alpha.-reductase. Petrow, V., et al., (1981), Steroids 38:121-140.
Other steroid 5-.alpha.-reductase inhibitors also have been described. U.S. Pat. No. 4,361,578 which issued Jun. 2. 1986, describes a class of homosteroid enzyme inhibitors. U.S. Pat. No. 4,191,759 discloses amides of 17.beta.-carboxy-4-androsten-3-one that are active as steroid 5-.alpha.-reductase inhibitors. Japanese Patents J60146855-A and J60116657-A disclose various aniline derivatives having numerous activities including 5-.alpha.-reductase inhibiting activity. Japanese Patent I60142941-A discloses phenyl-substituted ketones having 5-.alpha.-reductase inhibiting activity and European Patent EP173516-A discloses various phenyl-substituted amides having similar activity. Shiseido referenced terpene derivatives that are active inhibitors of steroid 5-.alpha.-reductase. Japanese Patent J59053417-A.
Recently it has been shown that steroid 5-.alpha.-reductase follows an ordered kinetic mechanism (Houston et al., (1987), Steroids 49: 355-369; Metcalf, B., et al., (1989) Bioorganic Chemistry 17:372-276) in which the nicotinamide confactors are the first substrate binding to the enzyme and the last product released from the enzyme (FIG. 1 ). Dead-end inhibitors of bisubstrate enzymes which follow ordered kinetic mechanisms can associate to one or more existing enzyme species. With steroid 5-.alpha.-reductase, enzyme forms to which such a steroidal-inhibitor might bind would include free enzyme, E, and enzyme binary complexes with cofactor, E-NADPH and E-NADP.sup.+. For example, that 4-aza steroidal inhibitors, such as N-t-butyl -5-.alpha.-androst- 1-ene-4-aza-17.beta.-carboxamide-3-one (compound A) (Liang, et al., (1985), Endocrinology 117:571-579), inhibit enzyme activity by forming an enzyme-NADPH-inhibitor dead-end complex (E-NADPH-1, FIG. 1).
In this context, the binding of inhibitors to each of these enzyme forms would be kinetically distinct as described in Irwin H. Segal, Enzyme Kinetics, Pub: John Wiley & Sons, Inc. (1975). The response of the velocity of enzyme catalysis with varying concentrations of testosterone in the presence of an inhibitor that binds to the E-NADPH complex can be described by equation 1; this model is denoted as competitive inhibition versus the variable substrate, in this case testosterone. Similarly, the model for an inhibitor that preferentially associates to the E-NADP.sup.+ complex would be described by equation 2; such a compound is referred to as an uncompetitive inhibitor versus the variable substrate. In equations 1 and 2, v is the observed velocity of product formation, V.sub.m is the maximal enzyme velocity at saturating concentrations of the variable substrate (A), I is the concentration of the inhibitor with apparent inhibition constants of K.sub.is or K.sub.ii, and K.sub.a is the apparent Michaelis constant for the variable substrate. EQU v=V.sub.M A/[K.sub.a (1+I/K.sub.is)+A] (1) EQU v=V.sub.M A/[K.sub.a +A(1+K/K.sub.ii)] (2)
Velocities (v) determined at variable concentrations of substrate (A) and inhibitor (I) are evaluated by non-linear curve fittings with computer programs as described by Cleland (Cleland, W. W. (1979) Methods in Enzymology 63, 103-138), to determine the best fit to equations 1 or 2. The results of these analyses are typically expressed in double reciprocal plots: 1/velocity versus 1/[testosterone]. The patterns in FIG. 1 are characteristic of a competitive and a uncompetitive inhibitor versus the second substrate in an ordered kinetic mechanism, and can be used to distinguish between the binding of a reversible dead-end inhibitor to E-NADPH or E-NADP.sup.+, respectively. Since the same uncompetitive model (equation 2) is used to describe an inhibitor that binds to either E-NADPH or E-NADP.sup.+ upon variation of the first substrate which binds to the surface of an ordered-enzyme, discrimination between these two mechanisms would not be possible by such an experiment with varying NADPH; the results from such an experiment can be used, however, to discriminate from a mechanism of inhibition resulting from binding to free enzyme (E).
The effects of inhibitors which act by these two differing mechanisms under comparable conditions are shown in FIG. 2; in this example, the concentration of each inhibitor has been set to be equal to its inhibition constant (K.sub.ii or K.sub.is) and the Michaelis-constant for substrate (K.sub.m) has been set a 1 concentration unit. Curves B and C represent inhibitors that preferentially bind to the E-NADPH and E-NADP.sup.+ complexes while curve A represents the uninhibited velocity. Note that curves A and B intersect on the ordinate (competitive pattern) while curves A and C are parallel (uncompetitive pattern). From this plot, it can be seen that a competitive inhibitor (E-NADPH-I) is more efficient at low substrate concentration, while the uncompetitive inhibitor (E-NADP.sup.+ -J) is more efficient at higher concentrations of substrate. The intersection point for these two curves occurs at the concentration of substrate that equal its K.sub.m.
A single molecule that could bind equally well to both E-NADPH and E-NADP.sup.+ would demonstrate the additive effects in slope and intercept of the two double-reciprocal plots. This is described by equation 3, and is represented by carve D in FIG. 2. EQU v=V.sub.m A/[K.sub.a (1+I/K.sub.is)+A(1+I/K.sub.ii)] (3)
Inhibition by a molecule that demonstrates such a mixed (noncompetitive) mode of action is more efficient that either of the single mechanisms alone over the entire substrate concentration range. This description of a mixed mode interaction is equally applicable to inhibition of steroid 5-.alpha.-reductase in the presence of two different molecular species which interact independently with the different enzyme forms, E-NADPH and E-NADP.sup.+. Thus, curve D also describes the inhibition by two compounds, each at concentrations equivalent to their inhibition constants for the E-NADPH and E-NADP.sup.+ complexes. Again, the result is superior to that of the single inhibitor throughout the entire concentration range of substrate.
Such additive inhibitory effects of a competitive inhibitor (N-t-butyl-5-.alpha.-androst -1-ene-4-aza-17.beta.-carboxamide-3-one, compound A) and an uncompetitive inhibitor (N,N-diisopropyl-androst-3,5-diene-17.beta.-carboxamide-3-carboxylic acid, compound B) have been demonstrated in vitro with double inhibition experiments; increasing concentrations of one inhibitor in the presence of the second induces greater enzyme inhibition (FIG. 3), while also demonstrating mutually exclusive binding. For a given concentration of one of the inhibitors, which binds to ENADPH (competitive) or E-NADP.sup.+ (uncompetitive), supplementation with any amount of the second would increase the observed enzyme inhibition over that of the reference inhibitor alone.
In comparison, FIG. 4 represents a model incorporating conservation of drug substance. Here, the calculated curves are based on the total chug substance relative to its respective inhibition constant: total inhibitor=constant=.SIGMA.([inhibitor]/K.sub.i,app). Curve A represents the uninhibited velocity, curves B and C represent the presence of only competitive or uncompetitive inhibitors, respectively, while curve D presents conservation of inhibitor substance composed of half competitive and uncompetitive inhibitors. Here, combination of inhibitors which function by the two different mechanisms is superior to the competitive inhibitors if the substrate concentration exceeds its K.sub.m, and is more efficient than the uncompetitive inhibitors at low substrate concentration below K.sub.m. A similar analysis versus the other substrate, NADPH, would demonstrate no difference in inhibition efficiency since both kinetic models are described by equation 2.
No prior art of combining 5-.alpha.-reductase inhibitors of different mechanistic types into a pharmaceutical composition to achieve superior results is known to the applicants.